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Open AcceResearch articleTissue MicroArray (TMA) analysis of normal and persistent Chlamydophila pneumoniae infectionNicole Borel*†1, Sanghamitra Mukhopadhyay†2,5, Carmen Kaiser1, Erin D Sullivan2, Richard D Miller3, Peter Timms4, James T Summersgill2,3, Julio A Ramirez2 and Andreas Pospischil1

Address: 1Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland, 2Division of Infectious Diseases, Department of Medicine, University of Louisville, Louisville, Kentucky, USA, 3Department of Microbiology and Immunology, University of Louisville, Louisville, Kentucky, USA, 4Infectious Diseases Program, School of Life Sciences, Queensland University of Technology, Brisbane, Australia and 5Biological Defense Research Directorate, Naval Medical Research Center, 12300 Washington Avenue, Rockville, MD 20852, USA

Email: Nicole Borel* - n.borel@access.unizh.ch; Sanghamitra Mukhopadhyay - Mukhopadhyays.ctr.in@nmrc.navy.mil; Carmen Kaiser - c.kaiser@access.unizh.ch; Erin D Sullivan - edsull01@gwise.louisville.edu; Richard D Miller - rdmill01@louisville.edu; Peter Timms - p.timms@qut.edu.au; James T Summersgill - j.summersgill@louisville.edu; Julio A Ramirez - jarami01@gwise.louisville.edu; Andreas Pospischil - apos@vetpath.unizh.ch

* Corresponding author †Equal contributors

AbstractBackground: Chlamydophila pneumoniae infection has been implicated as a potential risk factor foratherosclerosis, however the mechanism leading to persistent infection and its role in the diseaseprocess remains to be elucidated.

Methods: We validated the use of tissue microarray (TMA) technology, in combination withimmunohistochemistry (IHC), to test antibodies (GroEL, GroES, GspD, Ndk and Pyk) raisedagainst differentially expressed proteins under an interferon-gamma (IFN-γ) induced model ofchlamydial persistence.

Results: In the cell pellet array, we were able to identify differences in protein expression patternsbetween untreated and IFN-γ treated samples. Typical, large chlamydial inclusions could beobserved in the untreated samples with all antibodies, whereas the number of inclusions weredecreased and were smaller and atypical in shape in the IFN-γ treated samples. The staining resultsobtained with the TMA method were generally similar to the changes observed between normaland IFN-γ persistence using proteomic analysis. Subsequently, it was shown in a second TMAincluding archival atheromatous heart tissues from 12 patients undergoing heart transplantation,that GroEL, GroES, GspD and Pyk were expressed in atheromatous heart tissue specimens as well,and were detectable morphologically within lesions by IHC.

Conclusion: TMA technology proved useful in documenting functional proteomics data with themorphologic distribution of GroEL, GroES, GspD, Ndk and Pyk within formalin-fixed, paraffin-embedded cell pellets and tissues from patients with severe coronary atherosclerosis. Theantibodies GroEL and GroES, which were upregulated under persistence in proteomic analysis,displayed positive reaction in atheromatous heart tissue from 10 out of 12 patients. These may beuseful markers for the detection of persistent infection in vitro and in vivo.

Published: 19 October 2006

BMC Infectious Diseases 2006, 6:152 doi:10.1186/1471-2334-6-152

Received: 01 June 2006Accepted: 19 October 2006

This article is available from: http://www.biomedcentral.com/1471-2334/6/152

© 2006 Borel et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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BackgroundChlamydophila (C.) pneumoniae is an obligate intracellularpathogen which causes both acute and chronic respiratoryinfections in humans [1-5]. Over the last decade, severalreports in the literature have suggested that infection withC. pneumoniae may also contribute to the pathogenesis ofatherosclerosis [6,7]. C. pneumoniae was detected inatheromatous lesions by isolation in pure culture,polymerase chain reaction (PCR), electron microscopy, insitu hybridization (ISH) and immunohistochemistry(IHC) [8-11]. In order to play a causative role in chronicdiseases, C. pneumoniae would need to persist withininfected tissue for extended periods of time, thereby stim-ulating a chronic inflammatory response.

In vitro, an alteration of the normal developmental cycleof C. pneumoniae can be induced by interferon-γ-mediatedinduction of the host cell indoleamine 2,3-dioxygenase(IDO) activity, leading to a persistent form of the organ-ism [12-15]. In addition, several other models of in vitropersistence have been described (i.e. iron-limitation andantibiotics) [16]. Nevertheless, it is unknown which genesand proteins of C. pneumoniae are involved in the develop-ment and maintenance of persistence. We have previouslycharacterized an IFN-γ induced model of persistence by2D gel electrophoresis [17-19], and identified several pro-teins that are differentially regulated during the inductionof persistence.

Tissue microarray (TMA) technology, developed byKononen et al., 1998 [20] represents a promisingapproach in the field of proteomics for its potential use-fulness in in situ analysis. Preparations for TMA are con-structed by obtaining cylindrical tissue specimens fromparaffin blocks, assembling several hundreds into a singleblock, and preparing sections containing multiple tissuespecimens [20-22]. TMA sections can be analyzed usingstandard pathology methods, such as hematoxylin andeosin (HE) staining or special stains and in situ analyses,such as immunohistochemistry (IHC) [20,21,23-25]. Theutility of TMA protocols for high-throughput expressionprofiling of tumors at the molecular and protein levels hasbeen widely used in human cancer research on formalin-fixed and paraffin-embedded biopsy specimens[20,21,26,27].

Since many markers of gene and protein expression arefirst established and studied in cell culture systems, it isuseful to include cultured cells in TMAs for preliminarystudies when translating experimental techniques fromlaboratory systems to studies of human tissue. Therefore,in the present study, we used 5 polyclonal antibodiesdirected against differentially regulated chlamydial pro-teins during in vitro persistence [18,19] to validate the useof TMA technology on non-persistently, persistently

infected and uninfected HEp-2 cell pellets. In addition,archival atheromatous heart tissues [10] were tested byTMA, in combination with IHC, using the same antibod-ies, to determine their potential future use in detectingpersistently infected tissue.

MethodsCell lineHEp-2 cells (ATCC CCL-23) were obtained from theAmerican Type Culture Collection (Rockville, MD) andmaintained in Iscove's Maintenance Medium (IMM)(Cellgro, Washington, DC), as described previously [28].

Bacterial isolateC. pneumoniae A-03 (ATCC VR-1452), previously isolatedfrom an atheroma of a patient with coronary artery dis-ease during heart transplantation at the Jewish HospitalHeart and Lung Institute, Louisville, KY [10], were propa-gated in HEp-2 cell monolayers in Iscove's GrowthMedium (IGM), as described previously [28]. Elementarybodies (EBs) were harvested and purified by disruption ofHEp-2 cell monolayers with a cell scraper, sonication andcentrifugation over a renografin density gradient [28]. EBsuspensions were stored in sucrose-phosphate-glutamicacid buffer at -80°C, after which viable titers were estab-lished using standard methods.

Patients specimensArchival atheromatous tissue specimens from twelvepatients undergoing heart transplantation were investi-gated. Results from culture, PCR, IHC, ISH, EM and serol-ogy testing have been described previously [10]. The studyof Ramirez et al. was approved by the Institutional ReviewBoards (IRB) at both the University of Louisville and Jew-ish Hospital Healthcare Corporation.

Preparation of antibodiesFive proteins were identified as being differentially regu-lated in the IFN-γ-induced model of persistence [17,19]:(i) GroEL (60 kDa chaperonin) and (ii) GroES (10 kDachaperonin) are both chaperons involved in protein fold-ing, assembly and modification, (iii) GspD (general secre-tion protein D) involved in general protein secretion, (iv)Ndk (nucleoside-2-phosphate-kinase) involved in baseand nucleotide metabolism of amino acid biosynthesis,and (v) Pyk (pyruvate kinase) involved in the energymetabolism (glycolysis and gluconeogenesis). GroEL wasanalysed and quantitated previously [17], and the remain-ing four proteins were selected and confirmed in an iden-tical fashion [19]. GroEL and GroES were upregulatedunder persistence, whereas GspD and Pyk remainedunchanged and Ndk was downregulated. Each proteinwas plugged from a silver-stained 2D gel, which wasobtained from a purified EB preparation [18,19,28], andused for antibody production of polyclonal antibodies at

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Harlan Bioproducts for Science, Inc. (Indianapolis, IN,USA). Pathogen-free, barrier-raised New Zealand whiterabbits were immunized four times with the antigens, andsera from the final bleed were used in this study. The spe-cificity of each antibody was confirmed in our laboratoryby 2D gel electrophoresis of a purified C. pneumoniae EBpreparation followed by western blot analysis, demon-strating specific reactivity on the blot which correspondedto the molecular weight and iso-electric point of eachindividual protein (data not shown).

Other antibodies usedThe following antibodies were also used in these studies.

(i) Chlamydiaceae family-specific mouse monoclonal anti-body directed against the chlamydial lipopolysaccharide(mLPS; Clone ACI-P, Progen, Heidelberg, Germany).

(ii) Chlamydiaceae family-specific rabbit polyclonal anti-body directed against both the chlamydial LPS and thechlamydial major outer membrane protein (MOMP)(pLPS/MOMP; Cygnus Technologies, Inc., Southport,NC).

TMA analysisInfection protocolHEp-2 cells were grown in 75-cm2 cell culture flasks (Cos-tar, Cambridge, MA) to confluency and inoculated withpurified C. pneumoniae EB (1 × 109 IFUs/flask) in IGMwith or without human recombinant IFN-γ (50 and 100 Uper ml), followed by centrifugation at 675 × g (Sorvall TR6000D) for 30 min at 10°C and incubating at 37°C in 5%CO2 for 24, 48 and 72 hpi. After the respective incubationperiod, the medium was aspirated and the monolayerswere washed twice with 1 × phosphate buffer saline (PBS).Monolayers were fixed with 4% formalin in 1 × PBS for 10min followed by two washes with 1 × PBS. All monolayerswere harvested from the flasks with a cell scraper andtransferred into 15 ml of 1 × PBS. After centrifugation at675 × g for 10 min at 20°C, the supernatant was dis-carded. The pellets were re-suspended in 5% BSA preparedin 1 × PBS and transferred to an Eppendorf™ tube (Eppen-dorf-Netheler-Hinz GmbH, Hamburg, Germany) withone drop of hematoxylin for visualization. The cell sus-pensions were centrifuged for 5 min at 950 × g and thesupernatant was discarded. The resulting pellets were thenembedded in paraffin using the Cytoblock™ cell blockpreparation system (Shandon™, Pittsburg, USA). For eachcondition (timepoints 24, 48 and 72 h, concentrations of50 U/ml and 100 U/ml of IFN-γ) four sets of infectedmonolayers were prepared. As controls, four sets of unin-fected HEp-2 monolayers without and with 100 U/mlIFN-γ, were prepared as pellets.

TMA setupTwo equal cell culture array blocks including four equalprepared sets of cell pellets were created with the TMABuilder from Histopathology Ltd., Hungary according tothe instructions of the manufacturer. Briefly, the recipientparaffin block with 24 holes of diameter 2 mm eacharranged in four columns and six rows was moulded withthe TMA Builder. The whole cell pellets from the donorblocks were punched out with the Paraffin-Punch-Extrac-tor and were arrayed in the preformed recipient paraffinblock according to protocol.

Formalin-fixed and paraffin-embedded coronary arteryspecimens were available from the 12 patients (1 to 7 par-affin blocks per patient). Three TMA blocks includingeach tissue of the 12 patients, an uninfected and aninfected control HEp-2 cell pellet (72 hpi, without IFN-γ)were created in an identical fashion. 4 μm slide were cutusing a standard microtome.

ImmunohistochemistryParaffin sections were stained with the following primaryanti-chlamydial-antibodies: (1) mLPS at a dilution of1:50, (2) pLPS/MOMP at a dilution of 1:400, (3) GroEL,GroES, GspD, Ndk and Pyk at a dilution of 1:200. Theseoptimal dilutions were previously determined. Detectionwas performed with the Detection Kit (Dako ChemMate™Detection Kit, Glostrup, Denmark) according to the man-ufacturer's instructions. Antigen retrieval was performedby one min enzyme digestion (Pronase) (mLPS and pLPS/MOMP) and microwave heating (750 W for 10 minutes)two times in citrate buffer (pH 6,0; Target Retrieval Solu-tion (× 10), Dako ChemMate™, Glostrup, Denmark)(GroEL, GroES, GspD, Ndk and Pyk), respectively. Forinhibition of the endogenous peroxidase activity, theslides were immersed in peroxidase-blocking solution(Dako ChemMate™, Glostrup, Denmark) for 5 min atroom temperature (RT). Two additional blocking solu-tions were added to the slides, which were incubated withthe polyclonal antibodies GroEL, GroES, GspD, Ndk andPyk: Dako Protein Block Serum-free for 5 min at roomtemperature (Dako ChemMate™, Glostrup, Denmark)and 20 min Avidin D solution followed by 20 min Biotinsolution at room temperature (Vector). The slides wereincubated with the primary antibody for 60 min (mLPSand pLPS/MOMP) or over night (GroEL, GroES, GspD,Ndk and Pyk) at room temperature in a moist chamber. Intotal, the IHC for each antibody was repeated at least fourtimes on consecutive sections.

ResultsCell pellet arrayResults with the monoclonal antibody directed againstLPS (mLPS) and the polyclonal antibody directed againstchlamydial LPS and MOMP (pLPS/MOMP) were similar.

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Typical large, uniformly-staining chlamydial inclusions,located near the host cell nucleus, could be seen at 48 and72 hpi in the untreated samples with the mLPS (Figure 1)and the pLPS/MOMP antibody. Results with the 5 poly-clonal antisera tested, showed similar staining patterns inthe untreated samples, however, not all inclusions werestained uniformly positive (Figure 1). Earlier in thechlamydial developmental cycle at 24 h, granular positivematerial was seen in the cytoplasm of untreated cell pel-lets with the mLPS antibody and the pLPS/MOMP anti-body, as well as the GroEL, GroES and Pyk antibodies, andto a lesser extent, with the GspD (data not shown). All pel-lets at 24 hpi were negative with the Ndk antibody.

The IFN-γ-treated samples displayed an overall decrease inpositive reactivity. At 48 and 72 hpi, the number of inclu-sions were decreased and were obviously smaller andatypical in morphology (Figure 1). This could be observedfor all antibodies, except for Ndk, which was negative inall IFN-γ-treated samples (Figure 1). There was no differ-ence in inclusion morphology and antibody staining pat-terns seen between the two concentrations of IFN-γ (50 U/ml or 100 U/ml). Overall, the assay showed good repro-ducibility in all four replicates of monolayers.

TMA with atheromatous heart tissuesGranular, positive-staining material was seen in the cyto-plasm of subintimal macrophages and smooth musclecells in the medial part of the affected coronary arteries ofall patients with at least one antibody, except the twopatients with negative results for the presence of C. pneu-moniae in the original study (patients # 7 and 11) [10].Patients # 10 and 12 were positive with all antibodiesused except of the Ndk, which was negative in all 12 hearttissue specimens. Patients # 1, 2, 3 and 9 were positivewith the GroEL, GroES, GspD and Pyk, whereas patients #4 and 6 were only positive with the GroEL, GspD and Pyk.Patient # 5 was positive with the GroES and Pyk, andpatient # 8 revealed positive reaction with the GroEL andGspD. All patients were negative when tested with themLPS, whereas the pLPS/MOMP ab revealed positive reac-tions in patients # 10 and 12.

Positive staining of the antibodies GroEL and GroES inpatient # 10 and GspD and Pyk in patient # 12, respec-tively, is displayed in figure 2.

DiscussionIn this study, TMA technology was useful in documentingfunctional proteomics data showing the morphologic dis-tribution of GroEL, GroES, GspD, Ndk and Pyk within for-malin-fixed, paraffin-embedded cell pellets andatheromatous heart tissues. GroEL, GroES, GspD, Ndkand Pyk, selected as differentially regulated proteins fromproteomic analysis, were expressed in C. pneumoniae-

infected, untreated and IFN-γ treated HEp-2 cells andatheromatous heart tissues and were detectable morpho-logically by IHC. In general, IHC allows the detection ofthe presence of an antigen in tissue sections, however, theintensity of antigen labelling does not correlate with theamount of antigen present. The IHC labelling evaluated inthis study, therefore, represents the presence or absence ofproteins, but does not reflect quantitative expression ofGroEL, GroES, GspD, Ndk, Pyk, mLPS and/or pLPS/MOMP.

By using cell pellet array, in combination with IHC, it waspossible to identify the number, size and morphology ofthe chlamydial inclusions. Numbers of inclusions at 48hpi and 72 hpi were decreased under IFN-γ-persistenceand the inclusions were smaller and atypical, as describedpreviously [15], however, the staining intensity remainedthe same as that seen in untreated cells.

The staining results obtained with the TMA method weregenerally similar to the changes observed between normaland IFN-γ persistence using proteomic analysis [17-19].For example, GroEL, which was upregulated in proteomicanalysis, displayed more positive reactions in the IFN-γtreated samples when compared to GspD, whichremained unchanged in the proteomic analysis. Likewise,Ndk reactivity remained negative in all IFN-γ-treated sam-ples by TMA, which corresponded to the downregulationobserved in the proteomic analysis.

In comparison to other methods, such as PCR, we wereable to localize and visualize the positive reactions withinthe atheromatous heart tissue by TMA, in combinationwith IHC. The TMA method allowed a comparisonbetween multiple cell pellets or tissue specimens on onemicroscope slide. The 2 mm punch, in combination withthe Tissue Microarray Builder allowed easy manipulationduring punching, facilitating a rapid preparation of thecell culture array block. The fact that the 2 mm punch cancause significant damage to the donor block was not ofimportance because each donor block contained only onesingle cell pellet that was entirely punched out and thewhole area of each pellet could be examined. Other punchsizes such as 0.6 mm and 1.2 mm cause less damage to theoriginal donor block and make it possible to array severalhundreds of specimens on one single block, however thecreation of this block is more laborious. The problem ofreduction of the amount of tissue analyzed from thewhole cell pellet to a disk, which may be not be represent-ative of the protein expression of the entire tissue speci-men, can be solved by performing the experiments inseveral-fold redundancy. For that reason, we created twoequal cell pellet blocks including four sets of equally pre-pared monolayers containing the three timepoints(treated and untreated cell pellets and negative controls).

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cell pellet arrayFigure 1cell pellet array. Photomicrographs of TMA preparations of whole cell pellets showing differential expression pattern of C. pneumoniae proteins under untreated (A) and 50 U/ml IFN-γ treated (B) conditions at 48 hpi. Representative monolayers are shown to depict distinct differences in morphology and size of inclusions, as they were smaller and atypical under IFN-γ-induced persistence in comparison to the untreated monolayers.

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Multiple sections of the two TMA blocks were cut andprobed with each antibody.

Multiple sections of 3 TMA blocks, including the athero-matous heart tissue, were investigated with each antibody.In our previous study, patients # 1, 2, 3, 6 and 12 werepositive by IHC using Chlamydia-specific and C. pneumo-niae-specific antibodies [10]. We were able to detect 5more positive patients with experimentally produced anti-GroEL, anti-GroES, anti-GspD and anti-Pyk antibodies.Thus, differentially regulated proteins by proteomic anal-ysis were expressed in C. pneumoniae-infected human

atheromatous heart tissue specimens and were detectablemorphologically within lesions by IHC. The stainingresults obtained with the TMA corresponded to the reac-tivity as determined by proteomic analysis, for example,GroEL and GroES, which were upregulated under persist-ence in proteomic analysis, were likewise positive in mostheart tissue specimens. Patients # 7 and 11, which under-went heart transplantation primarily due to myocarditis,rather than severe atherosclerosis (unpublished data), dis-played questionable positive results (GroEL, GroES, GspDand Pyk). As these 2 patients were in fifth decade of lifeand had elevated anti-C. pneumoniae titers in the microim-

TMA with atheromatous heart tissuesFigure 2TMA with atheromatous heart tissues. Photomicrographs of TMA preparations of atheromatous heart tissues of patient # 10 showing positive reaction within macrophages with GroEL (1) and with GroES (2) and in patient # 12 showing positive reaction within smooth muscle cells with GspD (3) and Pyk (4).

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munofluorescence assay [10], we tend to assume that theysuffered from atherosclerosis and were Chlamydia-infected as patients # 1–6, 10 and 12, but perhaps to alesser extent. Definitive demonstration of chlamydial par-ticles in patients # 7 and 11 by more sensitive techniques(i.e. ultrastructural studies) is in progress [29].

ConclusionOverall, GroEL and GroES tend to be useful markers todetect persistent infection in vitro and in vivo. In addition,GspD and Pyk antibodies gave similar reactivity, indicat-ing appropriate sensitivity and specificity, and may alsoallow the detection of C. pneumoniae in chronically-infected tissue. Antibody prepared against Ndk remainednegative in tissue specimens from all 12 archived athero-matous tissue specimens, which corresponded nicely tothe downregulation observed in proteomic analysis.These data represent the first thorough examination ofatheromatous tissue by experimentally produced antibod-ies against C. pneumoniae and may provide a useful tech-nique to further define the role of this organism inatherosclerosis and other chronic human diseases.

Competing interestsThe author(s) declare that they have no competing inter-ests.

Authors' contributionsNB carried out the TMA analysis and drafted the manu-script. SM performed the cell culture infections and the 2D gel electrophoresis. CK performed the IHC. EDS partic-ipated in the cell culture infections and 2 D gel electro-phoresis. RDM and PT participated in the coordination ofthe study and helped to draft the manuscript. JTS and APparticipated in the design of the study. JAR provided theatheromatous heart tissue specimens. All authors read andapproved the final manuscript.

AcknowledgementsWe would like to thank Dr. Urs Ziegler and Claudia Dumrese, from the Institute of Anatomy, University of Zurich, Switzerland, for providing C. pneumoniae-infected HEp2 cells. This work was supported by grants from the NHMRC (PT) and the National Institutes of Health (HL68874 and A151255) (JTS).

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